The invention described herein was made in the course of work funded by U.S. Public Health Service Grants HL-19259 and CA 10126.
This invention relates to a process for separating immunologically activated human B lymphocytes from non-activated lymphocytes. Identification of surface markers and functional differences among lymphocytes obtained from various species has led to classification schems defining T and B lymphocytes and related cell types. Various subpopulations of these lymphocytes have also been identified. Functional differences among these cells and their subclasses have been recognized which reflect proliferative and differentiative states of maturation both dependent and independent of extrinsic immunological stimulation.
Populations of human T lymphocytes have frequently been defined by their ability to form rosettes with sheep erythrocytes (SRBC) although no functional evidence exists that demonstrates unequivocally that all SRBC rosetting lymphocytes are T cells or that all T cells necessarily will form SRBC rosettes. It has been suggested that human T lymphocytes might alternatively be defined by formation of rosettes with rhesus monkey erythrocytes (RhMRBC). It has been reported that human T lymphocytes, but not B lymphocytes, granulocytes or monocytes rosetted RhMRBC.
It is known that subsequent to exposure of specifically reactive B cells to antigen (in an immune response, in vivo or in vitro), such exposed cells differentiate and proliferate going on to form plasma cells which syntheize and secrete large amounts of specific antibodies. In similar fashion, several polyclonal activators (e.g., Epstein-Barr virus, Pokeweed mitogen) induce differentiation and proliferation in several clones of B cells. This polyclonal response leads to cells secreting many immunoglobulin types with multiple, usually unclassified specificities.
Production of specific antibodies in vitro in long-term continuous cell cultures requires that a set of human B cells, activated by some immunological means as described above, be converted to a long-term growing (continuously proliferating) cell line as a first step. The process of converting these cells to continuously growing cell line using Epstein-Barr virus, has been described in copending U.S. patent application Ser. No. 868,604, filed Jan. 11, 1979. Any such conversion is a multi-clonal process. Therefore, preselection of specifically (by antigen) stimulated cells provide a means to greatly enhance the chances of establishing a human B cell line with desired specificity, i.e., producing antibody to the antigen used to stimulate the B cells initially. Consequently, the utility of the invention described herein is that it enables one to separate such antigen activated human B cells prior to virus transformation, or some other technique used to convert B cells to continuous cell lines, thus greatly increasing statistical chances of obtaining cell lines synthesizing antibody specific for the activated antigen.